This proposal focuses on the fundamental question of how neuronal growth cones are accurately and precisely guided to their targets. It has long been established that growth cone navigation depends on regulated changes in both F-actin and microtubule (MT) dynamics in response to external guidance cues. However, the mechanisms by which these cues bring about specific changes in growth cone MT dynamics are an unresolved issue in the field. This proposal takes aim at that void, by investigating the functions of two interacting microtubule 'plus-end tracking proteins' (+TIPs), TACC3 and XMAP215, and their regulatory mechanisms. These two +TIPs uniquely bind to the extreme end of the MT, in front of all known others, and their binding to MTs is regulated by phosphorylation. Based on our preliminary data, we hypothesize that major guidance cue signaling pathways converge on TACC3 and XMAP215 to control MT plus-end dynamics and steer the growth cone. We will test this in Xenopus laevis using an array of complementary cell-based and biochemical approaches. The specific aims are: Aim 1 - Test the hypothesis that TACC3 and XMAP215, in response to guidance cues, facilitate axon guidance through spatial regulation of MT polymerization in growth cones. We will use in vivo gene transfer and time-lapse imaging of live axons in the brain of Xenopus laevis embryos, as well as high-resolution quantitative imaging of MT and +TIP dynamics in cultured neurons grown on stripes of guidance cues, to test the hypothesis that TACC3 and XMAP215, in response to guidance cues, facilitate axon guidance through spatial regulation of MT polymerization in growth cones. Aim 2 - Determine how kinase signaling controls TACC3 and XMAP215 function during growth cone navigation. We will use small molecule modulators, phosphomutant forms of TACC3 and XMAP215, and quantitative imaging analysis of +TIP dynamics in cultured neurons, along with biochemical approaches, to discern the effects of kinase regulation on +TIP activity in growth cones, to test the hypothesis that discrete kinase signaling pathways modulate TACC3 and XMAP215 regulation of MTs. Aim 3 - Define how TACC3 and XMAP215 directly control MT dynamics in vitro and in vivo. We will use multi-wavelength TIRF microscopy with in vitro MT reconstitution assays, structured illumination microscopy (SIM), and quantitative analysis of MT dynamics in growth cones, to test the hypothesis that TACC3 promotes guidance-mediated growth cone steering by locally enhancing the ability of XMAP215 to drive MT polymerization, while reducing the ability of XMAP215 to bind the MT lattice and couple MTs with F-actin retrograde flow. The results of these Aims will reveal direct mechanistic links between guidance cue signaling and regulation of the only well-characterized MT polymerase (XMAP215) and its key targeting factor (TACC3), all within the context of growth cone steering. As such, this proposed work has the potential to bring long-needed mechanistic understanding to the question of how extracellular cues govern MT dynamics to steer growth cones during neurodevelopment.